Mesenchymal stem cells (MSCs) are one of somatic stem cells (also referred to as biogenic stem cells or tissue stem cells) that may be differentiated into mesenchymal cells, such as osteoblasts, adipocytes, myocytes, chondrocytes, neurons, and hepatocytes. MSCs are expected to be applied for treatments in regenerative medicine in which tissues are remodeled or repaired. Examples of such treatments include anti-inflammation and immunosuppression, tissue repair, improvement of blood flow by angiogenesis, and anti-senescence (anti-aging). MSCs are also expected to be applied for treatments of disorders that have had no cures conventionally.
Multipotent mesenchymal stem cells can be separated not only from patient's tissues, such as bone marrows, adipose tissues, synovial membranes, alveolar bones, and periodontal ligaments, but also from various cells, such as placentas, umbilical cord blood, and umbilical cords, and thus the height of a bioethical hurdle to be cleared in order to use mesenchymal stem cells is low. Also because oncogenicity of mesenchymal stem cells may be lower than that of ES cells or iPS cells, an early clinical application of mesenchymal stem cells are expected.
When mesenchymal stem cells are clinically applied as cell and tissue pharmaceuticals or cell and tissue medical devices, for example, when these are applied to autologous transplantation, the application includes the steps of separating cells from a patient body, culturing the cells ex vivo to proliferate them, and autografting the cells to the patient. In this case, it is important to secure effectiveness and safety of the cells returned to the patient without any transformations to cells having characteristics which are other than the object during the time of culturing and/or preserving. However, quality of mesenchymal stem cells is hard to be controlled because they tend to be easily differentiated or aged, and thus quality control of these cells is a large issue.
In order to facilitate proliferation of mesenchymal stem cells cultured ex vivo, various proliferation peptides have been investigated (Patent Documents 1 and 2). Also, it is reported that when specific growth factors and a fatty acid complex are added to a serum-free basic medium, cell proliferation is comparable to, or even beyond the case where a serum-added medium is used (Patent Document 3). Meanwhile, it has been known that mesenchymal stem cells are divided in a limited number, and cells proceed to be aged depending on the number of divisions. Even now, how many divisions to induce proliferation are allowed to assure effectiveness of, that is, quality in clinical applications of mesenchymal stem cells is still not confirmed.
Therefore, mesenchymal stem cells collected from a patient are strongly desired to be preserved with high quality without deterioration until they are used for treatments. Without limiting to mesenchymal stem cells, a preservation method in which cells are cryopreserved at a temperature of liquid nitrogen is commonly used. Freezing and thawing will lose activities and functions of cells, and thus a chemical such as dimethyl sulfoxide (DMSO) is added to a freezing medium in order to protect cells during processes of freezing and thawing. DMSO is highly membrane-permeable and lowers ice crystal formation in the process in which cells are frozen to minimize damage of membranes and dehydration of organelles (Patent Document 4).
As an approach to preserve mesenchymal stem cells other than the freezing method, the cells may be preserved as cultured normally without freezing. However, the problem is that natures and characteristics of mesenchymal stem cells may be changed under a culture environment.
Generally, extracellular matrices constituting a surrounding environment of cells in vivo are known to chemically and physically affect differentiation states of connective tissue cells, and are reported to also affect determination of lineages that mesenchymal stem cells are differentiated into.
For example, induction of mesenchymal stem cells to be differentiated into neurologic, myogenic, or osteogenic cells by culturing the mesenchymal stem cells on a hydrogel having elasticity controlled to be similar to that of an in vivo biological environment has been investigated (Non-Patent Document 1). This finding means that mesenchymal stem cells will differentiate into lineages depending on characteristics of elasticity and the like in a culture environment, only by culturing the cells on a hydrogel substrate having, an elastic, modulus of 1 kPa or above, which means that the undifferentiated state of the mesenchymal stem cells is collapsed, and a quality of the cells will be deteriorated.
Meanwhile, a method in which mesenchymal stem cells are cultured on a type I collagen and fibronectin-coated acrylamide gel having elasticity of 250 Pa in order to introduce or keep the cell cycle of the stem cells in a static state to maintain biological activities of the cells, has been proposed (Non-Patent Document 2 and Patent Document 5).